The resistance to antibiotics developed by an increasing number of microorganisms is recognised to be a worldwide health problem (Tunger et al., 2000, Int. J. Microb. Agents 15:131-135, Jorgensen et al., 2000, Clin. Infect. Dis. 30:799-808). Thus, the development of non-antibiotic approaches for killing microorganisms is urgently required for controlling antibiotic-untreatable infections and limiting the development of additional antibiotic-resistant strains.
The treatment of microbial infections by photodynamic therapy (PDT) represents a valuable alternative method for eradicating bacteria since it involves a mechanism which is markedly different from that typical of most antibiotics. Thus, PDT is based on the use of a photosensitising molecule that, once activated by light, generates oxygen reactive species that are toxic for a large variety of prokaryotic and eukaryotic cells including bacteria, mycoplasmas and yeasts (Malik et al., 1990, J. Photochem. Photobiol. B Biol. 5:281-293; Bertoloni et al., 1992, Microbios 71:33-46). Importantly, the photosensitising activity of many photodynamic agents against bacteria is not impaired by the resistance to antibiotics but, instead, depends mainly on their chemical structure (Malik et al., 1992, J. Photochem. Photobiol. B Biol. 14:262-206).
Various types of neutral and anionic photosensitising agents exhibit a pronounced phototoxic activity against Gram positive bacteria. However, such photosensitising agents exert no appreciable cytotoxic activity against Gram negative bacteria unless the permeability of the outer membrane is altered by treatment with ethylene diamine tetra-acetic acid (EDTA) or polycations (Bertoloni et al., 1990, FEMS Microbiol. Lett. 71: 149-156; Nitzan et al., 1992, Photochem. Photobiol. 55:89-97). It is believed that the cellular envelope of Gram negative bacteria, which is more complex and thicker than that of Gram positive bacteria, prevents an efficient binding of the photosensitising agent or intercepts and deactivates the cytotoxic reactive species photogenerated by the photosensitising agent (Ehrenberg et al., 1985, Photochem. Photobiol. 41:429-435; Valduga et al., 1993, J. Photochem. Photobiol. B. Biol. 21:81-86).
In contrast, positively charged (cationic) photosensitising agents, including porphyrins and phthalocyanines, promote efficient inactivation of Gram negative bacteria without the need for modifying the natural structure of the cellular envelope (Merchat et al., 1996, J. Photochem. Photobiol. B. Biol. 32:153-157; Minnock et al., 1996, J. Photochem. Photobiol. B. Biol. 32:159-164). It appears that the positive charge favours the binding of the photosensitising agent at critical cellular sites that, once damaged by exposure to light, cause the loss of cell viability (Merchat et al., 1996, J. Photochem. Photobiol. B. Biol. 35:149-157). Thus, it has been reported that Escherichia coli is efficiently inactivated by visible light after incubation with the cationic 5,10,15,20-tetrakis-(4-N-methylpyridl-)-porphine (T4MPyP) (Valduga et al., 1999, Biochem. Biophys. Res. Commun. 256:84-88). The phototoxic activity of this porphyrin is mainly mediated by the impairment of the enzymic and transport functions of both the outer and cytoplasmic membranes, rather than by binding to DNA.
However, the utility of known porphyrin-based photodynamic therapy agents is limited due to their toxicity against mammalian host tissue cells, i.e. the compounds are unable to differentiate between target microbial cells and host cells. In addition, the utility of known porphyrin-based photodynamic therapy agents if further limited by their relatively low potency for target microbial cells.
Hence, there is a need for porphyrin-based compounds with improved toxicity profiles and high potency, which can be used in PDT to preferentially kill microbial cells.